Gene-Deleted Variant Strain Of Porcine Pseudorabies Virus, Vaccine Composition, Method Of Making The Same And Use Thereof

ABSTRACT

The present invention provides an attenuated strain of porcine pseudorabies virus (PRV), in which said attenuated strain of PRV is a variant strain of PRV with inactivation of gI/gE/11K/28K proteins. In addition, the present invention also provides a vaccine composition comprising the attenuated strain of PRV as an antigen, a preparation method and use thereof. Proved by immunogenicity and pathogenicity testing of the live vaccine, said live PRV vaccine can provide a good protection for pigs with no clinical signs observed, indicating excellent immune protection.

This application is a continuation of U.S. Patent Application No.14/901981, filed Dec. 29, 2015, which is a National Stage ofPCT/CN2015/070221, filed Jan. 6, 2015, which claims the benefit ofpriority to CN 201410418379.8, filed Aug. 22, 2014.

FIELD OF THE INVENTION

This invention relates to a gene-deleted variant strain of porcinepseudorabies virus, a vaccine composition prepared therefrom, a methodof making the same and a use thereof, belonging to the field of animalvirology.

BACKGROUND

Pseudorabies, also called Aujeszky's disease, is an acute infectiousdisease caused by Suid herpesvirus 1 (SuHV1) belonging to theAlphaherpesvirinae subfamily for many kinds of livestock such as swine,cattle and sheep, as well as poultry and wild animals, with the mainsymptoms of fever, intense itching (except swine) and encephalomyelitis.Pseudorabies in swine is found nationwide in China causing severedamages, and is one of the major diseases limiting the large-scaleproduction of pig farms. Infection can result in abortion, stillborn ormummified fetuses in pregnant sows, and neurological signs, paralysisand a high death rate in piglets. Pseudorabies virus (PRV) with strongpantropic properties, neurotropic properties and latent infectivity, mayestablish long-term latent infection in the peripheral nervous system,and then the latently infected host starts to get sick when the latentvirus is activated into the infectious virus.

According to recent researches, there are reports of new features ofpseudorabies, of which the significant manifestations include thatinfection among swine at any ages, horizontal transmission among swineherds, short incubation period (1˜2 days), morbidity rates between10%˜100%, mortality rate in pigs between 10%˜100% (mortality rate inpiglets can reach up to 100%), high fever in pigs after being infected(40° C.˜42° C., lasting for more than 3 days), dyspnea, diarrhea,wheezing, coughing, sneezing, hind limb paralysis, dog sitting, suddenlyfalling down, convulsions, lying on their sides, opisthotonus, makingstrokes with their arms, and finally dying of exhaustion, and theinfection also can cause reproductive disorder symptoms such as declinedsemen quality of boar, as well as abortion of pregnant sow (the abortionrate can reach up to 35%), premature birth, stillbirth, weakened piglets(weakened piglets die by 14 days of age), etc. By means of prior art,vaccinated pigs cannot completely resist attacks by the wild virus, andstill have symptoms like high fever, depression, partially or completelyloss of appetite, with a infection rate of more than 30% and a mortalityrate between 10% and 20%. According to literatures in the prior art, forexample, Jin-mei Peng, et al., Identification and antigenic variation ofnew epidemiology of pseudorabies virus from swine. Chinese Journal ofPreventive Veterinary Medicine, 2013, 35 (1):1-4; Tong Wu et al.,Identification and Characterization of a pseudorabies virus isolatedfrom a dead piglet born to vaccinated sow. Chinese Journal of Animalinfectious diseases, 2013, 21 (3):1-7; Yu et al., PathogenicPseudorabies Virus, China, 2012. Emerging infectious Diseases. 2014, 20(1):102-104; An et al., Pseudorabies virus variant inBartha-K61-vaccinated pigs, China, 2012. Emerging infectious Diseases.2013. 19 (11): 1749-1755, there is no vaccines capable of solving thepseudorabies caused by variant strains of porcine pseudorabies virus inthe prior art.

The Chinese patent application CN103756977A has disclosed a gE and gIdeleted variant strain of porcine pseudorabies virus, PRV-ZJ011G strain(of which the accession number is CGMCC No. 7957) and a vaccine preparedtherefrom, of which the content of virus prior to being inactivated is10^(6.0)TCID₅₀. Immunization with said vaccine could provide a 100%protection rate for 5 healthy piglets at 45 days of age. Said patentapplication, however, cannot successfully provide an attenuated livevaccine against new PRV strains, due to both humoral and cellularimmunity resulted from proliferation of attenuated live PRV vaccine invivo.

The Chinese patent application CN103981153A has disclosed theconstruction of a gene deleted variant strain of the pseudorabies viruslabeled with two fluorescent markers, wherein a variant strain ofpseudorabies virus with deletion of gE, gI, US9 and TK gene is insertedwith GRP gene and RFP gene as labels at the site of deleted genesthrough homologous recombination. Whereas in this patent applicationsaid a gene deleted variant strain is used as a vector of antigen, wedon't know if it has immunological competence itself, or how strong thevirulence of the variant strain is, since there are more than 70 genefragments for pseudorabies virus.

It has been disclosed by Chun-Hua Wang et al. (Chun-Hua Wang, Jin Yuan,Hua-Yang Qin, et al, A novel gE-deleted pseudorabies virus (PRV)provides rapid and complete protection from lethal challenge with thePRV variant emerging in Bartha-K61-vaccinated swine population in China,Vaccine 32 (2014)3379-3385) that 6-week-old piglets injected withPRVTJ-gE⁻ live vaccines got a complete protection from challenge anddidn't exhibit fever. Whereas as is known to those skilled in the art,the virulence of the PRV live vaccines depends on the age of piglets.Also according to the general experience in the art, the vaccines maynot be safe for 7-day-old piglets even though they are safe for6-week-old piglets. The live vaccines may spread among pigs, thereforethe vaccine which is only safe for 6-week-old piglets cannot be ensuredto be used clinically and a live vaccine which is safe for 7-day-oldpiglets, is required in the prior art to prevent effectively infectionwith the new variant strains of PRV.

SUMMARY OF INVENTION

In order to solve the deficiency of the prior art, the present inventionprovides an attenuated live strain of porcine pseudorabies virus forprevention and treatment of pseudorabies caused by mutated pseudorabiesvirus.

The main aim of present invention is to provide an attenuated strain ofporcine pseudorabies virus, wherein said attenuated strain of porcinepseudorabies virus is a pseudorabies virus strain with inactivation ofgI/gE/11K/28K proteins, preferably said PRV strain is a variant strainof PRV.

The inactivation of gI/gE/11K/28K proteins can be achieved by using wellknown methods in the art, including deletion of nucleotide sequenceexpressing the functional fragments of those above proteins from thegene, deletion of the whole ORF from the gene, or deletion or additionof one or more nucleotides whereby the gene cannot express functionalproteins normally or the proteins expressed don't have their originalfunction or have an extremely weak function.

As an embodiment of the present invention, the present inventionprovides an attenuated genetically engineered strain of porcinepseudorabies virus with deletion of gI/gE/11K/28K genes.

As an embodiment of the present invention, the whole ORF ofgI/gE/11K/28K genes was deleted from the genome of said attenuatedgenetically engineered strain of pseudorabies virus.

As an embodiment of the present invention, said variant strain ofpseudorabies virus is a virus strain of which gE protein has thesequence of SEQ ID NO. 5 or shares at least 95% homology to the sequenceof SEQ ID NO. 5; preferably, said variant strain of pseudorabies virusis obtained through isolation, and when infection with said variantstrain recurs in pigs previously immunized with attenuated gene-deletedstrain of pseudorabies virus according to the prior art, the pigs stilldisplay clinical signs of infection with said variant strain, selectedfrom high fever, depression and partial or complete loss of appetite;more preferably, said variant strain of pseudorabies virus is a variantstrain of pseudorabies virus and when infection with said variant strainrecurs in pigs previously immunized with attenuated strain of PRV withdeletion of one or more of gE, TK and gI genes, according to the priorart, the pigs are still infected with pseudorabies, which optionallycauses clinical signs of infection, selected from depression and loss ofappetite among piglets at the age of 9-10 days.

Most preferably, said variant strain of pseudorabies virus, includes,but are not limited to, PRV HN1201 strain (pseudorabies virus, strainHN1201)(deposited in the China Center for Type Culture Collection onMay, 20, 2013, of which the accession number is CCTCC NO. V 201311 andthe address is Wuhan University, Wuhan, China); JS-2012 strain (Wu Tong,Qingzhan Zhang, Hao Zheng et al. Isolation and identification of PRVfrom piglets infected after immunization [J]. Chinese Journal of AnimalInfectious Diseases. 2013, 21 (3): 1-7); PRV HeN1 strain (deposited inthe China General Microbiological Culture Collection Center on May, 20,2013, of which the accession number is CGMCC NO. 6656 and has beendisclosed in the patent application CN102994458A); NVDC-PRV-BJ strain,NVDC-PRV-HEB strain and NVDC-PRV-SD strain (Xiuling Yu, Zhi Zhou,Dongmei Hu,et al. Pathogenic Pseudorabies Virus, China, 2012 EmergingInfectious Diseases, www.cdc.gov/eid ol. 20, No. 1, January 2014); PRVHN1202 strain (pseudorabies virus, strain HN1202) (deposited in theChina Center for Type Culture Collection on Aug. 26, 2013, of which theaccession number is CCTCC NO. V 201335 and the address is WuhanUniversity, Wuhan, China); PRV TJ strain (Chun-Hua Wang Jin Yuan,Hua-Yang Qin, et al, A novel gE-deleted pseudorabies virus (PRV)provides rapid and complete protection from lethal challenge with thePRV variant emerging in Bartha-K61-vaccinated swine population in China.Vaccine. 32 (2014) 3379-3385); a variant strain of pseudorabies virusPRV-ZJ01 (with the accession number, CGMCC No. 8170, and disclosed inCN103627678A).

As an embodiment of the present invention, said PRV strain is HN1201strain, HN1202 strain, JS-2012 strain, PRV HeN1 strain, NVDC-PRV-BJstrain, NVDC-PRV-HEB strain or NVDC-PRV-SD strain, PRV TJ strain orPRV-ZJ01 strain.

As an embodiment of the present invention, said attenuated strain ofporcine pseudorabies virus is an attenuated strain of porcinepseudorabies virus with further inactivation of TK protein; preferablythe nucleotide sequence at the location of TK in the genome of saidattenuated strain of porcine pseudorabies virus encodes and expressesthe amino acid sequence shown in SEQ.NO. 4 of the sequence listing.

As a preferred embodiment of the present invention, the nucleotidesequence at the location of TK in the genome of said attenuated strainof porcine pseudorabies virus is the nucleotide sequence shown inSEQ.NO. 3 of the sequence listing.

As a preferred embodiment of the present invention, the presentinvention provides an attenuated genetically engineered strain ofporcine pseudorabies virus with deletion of gI/gE/11K/28K/TK genes.

As used herein, the term “variant strain of pseudorabies virus”, alsocalled highly pathogenic PRV strain, refers to diseases with significantmanifestations including infection among swine at any ages, horizontaltransmission among swine herds, short incubation period (1˜2 days),morbidity rates between 10%˜100%, mortality rate in pigs between10%˜100% (mortality rate in piglets can reach up to 100%), high fever ofpigs after being infected (40° C.˜42° C., lasting for more than 3 days),dyspnea, diarrhea, wheezing, coughing, sneezing, hind limb paralysis,dog sitting, suddenly falling down, convulsions, lying on their sides,opisthotonus, making strokes with their arms, and finally dying ofexhaustion, and reproductive disorder symptoms caused by infection suchas declined semen quality of boar, as well as abortion of pregnant sow(the abortion rate can reach up to 35%), premature birth, stillbirth,weakened piglets (weakened piglets die by 14 days of age), etc.Preferably, said variant strain of pseudorabies virus obtained throughisolation, and when infection with said variant strain recurs in pigspreviously immunized with attenuated gene-deleted strain of pseudorabiesvirus according to the prior art, the pigs still display clinical signsof infection with said variant strain, selected from selected from highfever, depression and partial or complete loss of appetite. Preferably,said variant strain of pseudorabies virus is a virus strain of which gEprotein has the sequence of SEQ ID NO. 5 or shares at least 95% homologyto the sequence of SEQ ID NO. 5. More preferably, said variant strain ofpseudorabies virus is a variant strain of pseudorabies virus wherein,when infection with said variant strain recurs in pigs previouslyimmunized with attenuated strain of porcine pseudorabies virus withdeletion of one or more of gE, TK and gI genes, according to the priorart, the pigs are still infected with pseudorabies, which optionallycauses clinical signs of infection selected from depression and loss ofappetite among piglets at the age of 9-10 days. The term “homology” inthe present invention refers to the level of similarity between twoamino acid sequences or two nucleotide sequences. The homology betweenamino acid sequences or nucleotide sequences can be calculated by anyappropriate methods well known in the art, for example, the target aminoacid (or nucleotide) sequence and the reference amino acid (ornucleotide) sequence is aligned, and gaps can be induced if necessary tooptimize the number of the identical amino acids (or nucleotides)between two aligned sequences, and the percentage of the identical aminoacids (or nucleotides) between two aligned sequences can be calculatedaccordingly. Alignment of amino acid (or nucleotide) sequences andcalculation of homology can be achieved by software well kwon in theart. Examples of such software include, but is not limited to, BLAST(which can be accessed through the website of the National Center forBiotechnology Information, NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgior can be found in Altschul S. F. et al, J. Mol. Biol, 215:403-410(1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)),ClustalW2 (which can be accessed through the website of the EuropeanBioinformatics Institute, EBI, http://www.eji.ac.uk/Toolsa/clustalw2/,or can be found in Higgins D. G. et al, Methods in Enzymology,266:383-402 (1996); Larkin M. A. et al, Bioinformatics (Oxford,England), 23 (21):2947-8 (2007)), and TCoffee (which can be accessedthrough the website of the Swiss Institute of Bioinformatics, SIB,http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi, or canbe found in, Poirot O. et al, Nucleic Acids Res., 31 (13):3503-6 (2003);Notredame C. et al, J. Mol. Biol, 302 (1):205-17(2000)) etc. It is allwithin the knowledge scope of a person skilled in the art that whenusing the software to do sequence alignment, he can use the defaultparameters provided by the software or adjust the parameters provided bythe software according to the actual condition.

The term “gI protein” is encoded by US7, and comprises 366 amino acids,with ORF located between 122298-123398.

The term “gE protein” is encoded by USB, and comprises 577 amino acids,with ORF located between 123502-125235.

The term “11K” is encoded by US9, and comprises 98 amino acids, with ORFlocated between 125293-125589.

The term “28K” is encoded by US2, and comprises 256 amino acids, withORF located between 125811-126581.

The term “TK”, also called thymidine kinase, is encoded by UL23, andcomprises 320 amino acids, with ORF located between 59512-60474.

The term “gI/gE/11K/28K” and “gI/gE/11K/28K/TK” in the present inventionrefers to “gI, gE, 11K and 28K” and “gI, gE, 11K, 28K and TK”,respectively, wherein “I” in the present invention refers to “and”, forexample, “inactivation of gI/gE/11K/28K proteins” refers to inactivationof gI, gE, 11K and 28K proteins.

Unless otherwise stated, the term “PRV-gI-gE-11K-28K-TK−” in the presentinvention refers to deletion of gI, gE, 11K, 28K and TK genes.

As a preferred embodiment of the present invention, said attenuatedgenetic strain of porcine pseudorabies virus with deletion ofgI/gE/11K/28K genes is attenuated genetically engineered virus strain ofPRV HN1201 strain with deletion of gI/gE/11K/28K genes.

As a preferred embodiment of the present invention, said attenuatedstrain of porcine pseudorabies virus includes HN1201 strain, HN1202strain, JS-2012 strain, PRV HeN1 strain, NVDC-PRV-BJ strain,NVDC-PRV-HEB strain or NVDC-PRV-SD strain, with deletion ofgI/gE/11K/28K.

As a preferred embodiment of the present invention, said attenuatedstrain of porcine pseudorabies virus is PRV HN1201 strain (pseudorabiesvirus, strain HN1201) with deletion of gI/gE/11K/28K genes using geneticengineering, wherein said PRV HN1201 strain is deposited in the ChinaCenter for Type Culture Collection on May, 20, 2013, of which theaccession number is CCTCC NO. V 201311 and the address is WuhanUniversity, Wuhan, China.

As used herein, the term “attenuated” in the present invention refersto: compared with unmodified parent strain, the virulence of thegene-deleted pseudorabies virus strain is reduced, of whichmanifestations include reduction of numbers of dead pigs, numbers ofpigs with fever, and duration of fever. If the statistically significantdifference of one or more parameters for determination of severity ofdiseases for virus strains decreases, its virulence is attenuated.

Another aspect of the invention relates to a vaccine composition,wherein said vaccine composition comprises an immune amount of antigenof said attenuated strain of porcine pseudorabies virus and carrier;preferably the content of antigen of the attenuated strain of porcinepseudorabies virus is not less than 10^(6.0)TCID₅₀/ml.

As a preferred embodiment of the present invention, the antigen of saidattenuated strain of porcine pseudorabies virus is live attenuatedstrain of porcine pseudorabies virus; said vaccine composition furthercomprises a cryoprotectant.

As an embodiment of the present invention, said vaccine compositioncomprises an immune amount of attenuated live vaccine of said variantstrain of pseudorabies virus with deletion of gI/gE/11K/28K genes andcarrier.

As a preferred embodiment of the present invention, said vaccinecomposition comprises an immune amount of attenuated live vaccine ofsaid variant strain of pseudorabies virus with deletion ofgI/gE/11K/28K/TK genes and carrier.

As a preferred embodiment of the present invention, said vaccinecomposition comprises an immune amount of attenuated live vaccine of avariant strain of pseudorabies virus with deletion of gI/gE/11K/28Kgenes, such as HN1201 strain, HN1202 strain, JS-2012 strain, PRV HeN1strain, NVDC-PRV-BJ strain, NVDC-PRV-HEB strain or NVDC-PRV-SD strain,PRV TJ strain or PRV-ZJ01 strain and carrier.

As a preferred embodiment of the present invention, said vaccinecomposition comprises an immune amount of attenuated live vaccine ofvariant strain of PRV strains with deletion of gI/gE/11K/28K/TK genes,such as HN1201 strain, HN1202 strain, JS-2012 strain, PRV HeN1 strain,NVDC-PRV-BJ strain, NVDC-PRV-HEB strain or NVDC-PRV-SD strain, PRV TJstrain or PRV-ZJ01 strain and carrier.

Preferably, the antigen of said attenuated strain of porcinepseudorabies virus is attenuated live PRV strain; said vaccinecomposition further comprises a cryoprotectant.

As an embodiment of the present invention, said vaccine composition isattenuated live vaccine of the PRV strain with deletion of gI/gE/11K/28Kgenes.

Optionally, one or more compounds with adjuvant activity may be added tovaccines. It does not necessarily require such an adjuvant to achievethe efficacy of the live attenuated pseudorabies virus according to thepresent invention, but especially for a combination vaccine comprisingthe live attenuated pseudorabies virus according to the presentinvention and antigenic materials from another pathogenic virus ormicroorganism (see below), it will be worth adding an adjuvant.Adjuvants are non-specific stimulators of the immune system. Theyimprove immune response of the host responding to a vaccine. Examples ofadjuvants known in the art is include complete/incomplete Freund'sadjuvant, vitamin E, non-ionic blocking copolymers, muramyl dipeptide,ISCOMs (immune stimulating complexes, refer to, for example the Europeanpatent EP 1099 42), saponins, mineral oil, vegetable oil, and Carbopol.

Therefore, in a preferred form of said embodiment, the live attenuatedvaccine according to the present invention further comprises anadjuvant.

Other examples of pharmaceutically acceptable carriers or diluents canbe used in the present invention, include stabilizers such as SPGA,carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose,dextran), proteins such as albumin or casein, protein-containing agentssuch as bovine serum or skimmed milk and buffers (e.g. phosphatebuffer).

Especially when such stabilizers are added to the vaccine, the vaccineis very suitable for freeze-drying. Therefore, in a more preferred formof said embodiment, the live attenuated vaccine is in a freeze-driedform.

In addition, said pseudorabies vaccine in the present invention can beused conjunctly with other inactivated pathogens or antigen to preparecombined vaccines or complex vacancies against various diseasesincluding pseudorabies. As used herein, the term “combined vaccine”refers to a vaccine prepared with the virus mixture by mixing thepseudorabies virus in the present invention with at least one differentvirus. The term “complex vaccine” refers to a vaccine prepared fromviruses and bacterium. For example, the pseudorabies virus in thepresent invention can be mixed or combined with classical swine fevervirus, porcine reproductive and respiratory syndrome virus, porcinecircovirus and/or haemophilus parasuis and mycoplasma.

As an embodiment of the present invention, said vaccine furthercomprises inactivated pathogens or antigen, preferably, said antigencomprises antigen of classical swine fever virus, antigen of porcinereproductive and respiratory syndrome virus, antigen of porcinecircovirus and/or haemophilus parasuis or antigen of mycoplasma.

As an embodiment of the present invention, the attenuated virus strainin the present invention can be inserted by exogenous genes, saidexogenous genes encode one or more antigens selected from a plurality ofpathogens infecting pigs, said pathogens consist of porcine reproductiverespiratory syndrome (PRRS) virus, porcine Influenza virus, porcineparvovirus, transmissible gastroenteritis virus, rotavirus, type 1 ortype 2 porcine circovirus virus, Escherichia coli, Erysipelothrixrhusiopathiae, Bordetella bronchiseptica, Haemophilus parasuis,Mycoplasma hyopneumoniae and Streptococcus suis.

Preferably, said vaccine composition may further comprise medium,adjuvants and excipients.

The vaccine composition according to the present invention may alsocomprises medium, adjuvants and/or excipients. Physiological saline ordistilled water can be used as medium.

Another aspect of the present invention relates to a method forpreparing said vaccine composition, wherein said method comprises thesteps: (1) said attenuated strain of porcine pseudorabies virus isamplified and cultured; and (2) a cryoprotectant is added into saidattenuated strain of porcine pseudorabies virus amplified and cultured.

Another aspect of the present invention relates to a use of said vaccinecomposition for preparing medicine for treatment and prevention ofpseudorabies.

As an embodiment of the present invention, said pseudorabies ispseudorabies caused by a variant strain of pseudorabies virus.

As used herein, the term “diseases relating to PRV” can further refer todiseases with significant manifestations including but not limited toinfection among swine at any ages, horizontal transmission among swineherds, short incubation period (1˜2 days), morbidity rates between10%˜100%, mortality rate in pigs between 10%˜100% (mortality rate inpiglets can reach up to 100%), high fever of pigs after being infected(40° C.˜42° C., lasting for more than 3 days), dyspnea, diarrhea,wheezing, coughing, sneezing, hind limb paralysis, dog sitting, suddenlyfalling down, convulsions, lying on their sides, opisthotonus, makingstrokes with their arms, and finally dying of exhaustion, andreproductive disorder symptoms caused by infection such as declinedsemen quality of boar, as well as abortion of pregnant sow (the abortionrate can reach up to 35%), premature birth, stillbirth, weakened piglets(weakened piglets die by 14 days of age), etc. The differences betweenabove described symptoms and symptoms caused by infection of regularpseudorabies virus in the prior art are: in adult pigs (whose weight isabove 50 kg), high fever of infected pigs (40° C.˜42° C., lasting formore than 3 days), dyspnea, diarrhea, wheezing, coughing, sneezing, hindlimb paralysis, dog sitting, suddenly falling down, convulsions, lyingon their sides, opisthotonus, making strokes with their arms, andfinally dying of exhaustion; sudden incidence of pseudorabies in newbornpiglets and piglets below the age of 4 weeks, further resulting inmassive death with a mortality of more than 90%; main manifestations ininfected piglets including increased body temperature over 41° C.,completely loss of appetite, obvious neurological signs and diarrhea;and in piglets just before or after being weaned, mainly respiratorysymptoms, such as dyspnea, coughing and runny noses, etc.

As used herein, the term “prevention” refers to all behaviors to inhibitthe infection of pseudorabies virus or delay the onset of the diseasevia administration of the vaccine composition according to the presentinvention. The term “treatment” refers to all behaviors to relieve orcure the symptoms caused by infection of PRV via administration of thevaccine composition according to the present invention.

ADVANTAGES OF THE PRESENT INVENTION

The strain in the present invention with less virulence could providebetter immune protection, induce an earlier generation of antibodies andthe challenge result after immunization shows that gE antibody is stillnegative.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing construction of plasmidpUCgI/gE/11K/28KA-GFP-B;

FIG. 2 is a schematic diagram showing the location for deletion ofgI/gE/11K/28K genes and location of homologous arms, USA and USB in thegenome;

FIG. 3 is a gel electrophoresis pattern for comparing PCR fragments ofPRV HN1201 strain before and after deletion of gI/gE/11K/28K genesthrough PCR method;

FIG. 4 is a schematic diagram showing the location for deletion of TKgene and location of homologous arms of TKA and TKB in the genome;

FIG. 5 is a gel electrophoresis pattern for comparing PCR fragments ofPRV HN1201 strain before and after deletion of TK gene through PCRmethod.

SEQUENCE LISTING

SEQ ID NO. 1 is the nucleotide sequence of gI/gE/11K/28K genes in thePRV HN1201 strain.

SEQ ID NO. 2 is the nucleotide sequence of TK gene in the PRV HN1201strain.

SEQ ID NO. 3 is the nucleotide sequence of location for deletion of TKgene in the PRV HN1201 strain.

SEQ ID NO. 4 is the amino acid sequence of location for deletion of TKgene in the PRV HN1201 strain.

SEQ ID NO. 5 is the amino acid sequence of gE in the PRV HN1201 strain.

DETAILED DESCRIPTION

The description of the present invention is further provided as followswith reference to the specific embodiments, and features and advantagesof the present invention will become more apparent from the followingdescription. However, these embodiments are only exemplary, but notforming any limitation to the scope of the present invention. It shouldbe understood by a person skilled in the art that modifications oralternatives to details and forms of the technical solution of thepresent invention without deviation from the spirit and scope of thepresent invention will be allowed, while those modification andalternatives should all fall within the scope of the present invention.

In the invention, the term “per pig” refers to the amount of vaccineeach pig injected.

In the invention, the term “TCID₅₀” refers to 50% tissue cultureinfective dose, a way to represent viral infectivity.

Minimum Essential Medium (MEM) liquid medium is prepared with MEM drypowdered medium purchased from Life Technologies, Corp. according to theinstruction.

Dulbecco's Modified Eagle's Medium (DMEM) in the present invention isprepared with reference to the preparation method from Appendix A ofGB/T18641-2002 Diagnostic Techniques for Aujeszk's Disease.

In the present invention, the term “PBS” is the abbreviation forPhosphate Buffer Saline, and 0.01 mM pH 7.4 PBS as used in the presentinvention is prepared as described in Molecular cloning: Laboratorymanuals, 3rd edition.

The PRV HN1201 strain (pseudorabies virus, strain HN1201) used in theembodiments is deposited in the China Center for Type Culture Collectionon May, 20, 2013, of which the accession number is CCTCC NO. V 201311and the address is Wuhan University, Wuhan, China.

The PRV HN1202 strain (pseudorabies virus, strain HN1202) used in theembodiments is deposited in the China Center for Type Culture Collectionon Aug. 26, 2013, of which the accession number is CCTCC NO. V 201335and the address is Wuhan University, Wuhan, China.

PRV is the abbreviation for the term Pseudorabies virus.

In the following specific embodiments, the description of the presentinvention is further provided with examples of PRV HN1201 strain,NVDC-PRV-BJ strain, NVDCPRV-HEB strain, NVDC-PRV-SD strain and HN1202strain.

EXAMPLE 1 Preparation of PRV HN1201 Strain With Deletion ofgI/gE/11K/28K 1.1 Construction of a Transfer Vector for Recombinant PRVHN1201GFP Virus

According to the sequence of US segment (gI/gE/11K/28K) to be deleted,the homologous arms were designed at its two ends, called USA and USB,respectively. USA and USB were cloned into pUC19 vector and namedpUCUSAB. Then GFP gene was cloned into pUCUSAB, to obtain a transfervector for recombinant virus which was called pUCUSA-GFP-B. Thehomologous arms in the transfer vector are sequences of two sides of US,therefore the recombinant virus obtained after recombination, was USsegment deleted, which comprised gI/gE/11K/28K. FIG. 1 is a schematicdiagram showing construction of the transfer vector, and FIG. 2 showsthe location of the homologous arms, USA and USB in the genome.

1.1.1 Amplification and Cloning of the Homologous Recombinant Arms1.1.1.1 Design of Primers and Preparation of Templates

Two pairs of primers were designed for amplifying the homologous arms attwo sides of segment to be deleted according to the gene sequence ofHN1201 virus:

The upstream and downstream primers for the homologous arm USA at theleft side are, respectively:

USAF: CCGGAATTCTCGTCGTGGGCATCGTCATCAT(the underline portion refers to the EcoR I cutting site) USAR:CTATCTAGAataacttcgtataatgtatgctatacgaagttat CGGTACTGCGGAGGCTACGGAC(the underline portion refers to the Xba Icutting site, lowercase letters refer to the loxp site

The upstream and downstream primers for the homologous arm USB at theright side are, respectively:

USBF: ACATGCATGCataacttcgtatagcatacattatacgaagttatACGGCAGGATCTCTCCGCGTCCC(the underline portion refers to the SphI cuttingsite, lowercase letters refer to the loxp site) USBR:CCCAAGCTTAGGAGGGGGCGGGGAGCGCGAGC(the underline portion refers to the Hind III cutting site)

Vero cells were transfected with PRV HN1201, and part of supernatant washarvested when the cytopathic effect of cells reached to 80%, forextracting genomic DNA of virus by using Geneaid Viral Nucleic AcidExtraction kit as the template for amplification of the homologous arms.

1.1.1.2 Amplification and Cloning of the Homologous Arms, USA and USB

USA and USB were amplified through PCR method by using TAKARA PrimeSTAR,of which the system and condition is as follows:

PRV HN1201 DNA 1 μL PrimeSTAR 0.5 μL 2*primeSTAR GC buffer 25 μL dNTP(25mM) 4 μL Upstream primer 0.5 μL downstream primer 0.5 μL Water Used foradjusting to a final volume of 50 μL

98° C. 2 min 98° C. 10 s 68° C. 1 min } 30 cycles 15 s 68° C. 5 min

USA and USB fragments amplified by PCR were separated by electrophoresison agarose gel, and the target fragments were recovered with TIANGEN GelRecovery Kit. USA fragment and pUC19 vector was digested by both of EcoRI and XbaI, and the target fragments were recovered, connected by T4 DNAligase, and the product was transformed into DH5α. The transformationmix was spread onto plates containing ampicillin, and incubated at 37°C. overnight. A single colony was picked to extract the plasmid and theplasmid was identified using enzyme digestion, and the correct plasmidafter identification was named pUCUSA. pUCUSA and USB was digested byboth of SalI and HindIII, and the target fragments were recovered,linked by T4 DNA ligase, and the product was transformed into DH5α. Thetransformation mix was spread onto plates containing ampicillin, andincubated at 37° C. overnight. A single colony was picked to extract theplasmid and the plasmid was identified by sequencing after enzymedigestion, and the correct plasmid after identification was namedpUCUSAB.

1.1.3 Amplification of Label Gene GFP 1.1.2.1 Removal of MultipleCloning Site (MCS) of GFP Vector pAcGFP-C1

The pAcGFP-C1 plasmid (purchased from Clontech, Catalog No. 632470) wasdigested by Bgl II and Sma I, and the linearized vector was recovered,linked by T4 DNA Ligase after filling-in with DNA Polymerase I Large(Klenow) Fragment, and transformed into the competent cell DH5α toobtain MCS deleted GFP plasmid, named pAcGFPΔMCS.

1.1.2.2 Amplification of GFP Gene

The primers for amplifying GFP were designed according to the sequenceof pAcGFP-C 1 vector.

Upstream primer CMVU: ACGCGTCGACTAGTTATTAATAGTAATCAATTACG(the underline portion refers to the SalI cutting site.)Downstream primer SV40R: ACATGCATGCCTAGAATGCAGTGAAAAAAATGC((the underline portion refers to the Sph I cutting site.)

GFP gene was amplified with pAcGFPΔMCS plasmid as the template, of whichthe system and condition is as follows:

pAcGFPΔMCS 1 μL primeSTAR 0.5 μL 2*primeSTAR GC buffer 25 μL dNTP(25 mM)4 μL Upstream primer CMVU 0.5 μL Downstream primer SV40R 0.5 μL WaterUsed for adjusting to a final volume of 50 μL

94° C. 2 min 94° C. 30 s 60° C. 30 s 30 cycles {close oversize brace}72° C. 2 min 72° C. 5 min

A target band was recovered by electrophoresis on agarose gel forfurther linking.

1.1.3 Linking of GFP Label Gene and pUCUSAB

GFP was digested with both of Sal and Sph I, and the target fragmentswere recovered, linked to pUCUSB plasmid which had been through the samedouble enzyme digestion, and the product was transformed into thecompetent cell DH5α. The transformation mix was spread onto platescontaining ampicillin, and incubated at 37° C. overnight. A singlecolony was picked to extract the plasmid and the plasmid was identifiedby sequencing after enzyme digestion, and the correct plasmid afteridentification was named pUCUSA-GFP-B.

1.2 Acquisition of Recombinant Virus Containing GFP 1.2.1 Acquisition ofRecombinant Virus Through Co-Transfection of Vero Cells With theTransfer Vector and HN1201 DNA

Co-transfection of vero cells was conducted by using lipofectintechnique, wherein 3 μg PRV-HN1201 viral genomic DNA and 5 μg thetransfer vector pUCUSA-GFP-B was transfected, in accordance withprocedures of Lipofectamine 2000 Protocol (Invitrogen, Catalog No.11668030). Cells were incubated at 37° C. in an incubator containing 5%CO₂. The supernatant of cell culture, i.e. P0 recombinant virus, namedrPRV-GFP-US−, was collected 36-48h after transfection, or until thecytopathic effect was visible and infected cells exhibited fluorescence.

1.2.2 Plaque Purification of Recombinant Viruses

When infected with the obtained PO recombinant virus rPRV-GFP-US−, verocells infected were covered with 2% agarose with low melting point.After 48h when the cytopathic effect became apparent and infected cellsexhibited obvious fluorescence, a plaque with a green fluorescence waspicked and freeze-thawed 3 times in −70° C., inoculated at 10-foldserial dilutions into vero cells previously laid in six-well plates.Such plaque with a green fluorescence was continued to be picked forpurification. After 8 rounds of plaques purification, a purifiedrecombinant virus rPRV-GFP-US−which was free of wild-type virus HN1201and with deletion of gI/gE/US9/US2 (i.e. gI/gE/11K/28K) was obtained.

1.3 Deletion of GFP Label Gene in the gI/gE/US9/US2 (i.e. gI/gE/11K/28K)Segment-Deleted Recombinant Virus

pBS185 plasmid expressing Cre enzyme (purchased from addgene, Cre enzymerecognizes loxP sites at downstream of USA and upstream of USB, whereinUSA and USB are homology arms, and deletes sequence between two loxpsites) and genomic DNA of recombinant virus rPRV-GFP-US− wasco-transfected into vero cells, with the results showing relativelyobvious cytopathic effect and more single fluorescence 24 h aftertransfection. After serial dilution, PO virus harvested was inoculatedfor plaque screening; fluorescence-negative plaque was picked for thenext round of purification. After 2 rounds of screening andpurification, a fluorescence-negative virus was obtained, and namedvPRV-US−. PCR identification result after extraction and purification ofviral genomic DNA, showed deletion of gI/gE/US9/US2 (i.e. gI/gE/11K/28K)segment, and indicated that GFP label gene had been deleted. The resultshowed a successful purification of gI/gE/US9/US2 (i.e. gI/gE/11K/28K)segment-deleted virus containing no GFP label gene.

1.4 Confirmation of PRV HN1201 Strain With Deletion of US Segment

The viral genome of gI/gE/US9/US2 (i.e. gI/gE/11K/28K) segment-deletedvirus and wild-type virus, was extracted and identified by PCR, with thefollowing primers:

USDCF: TACATCGTCGTGCTCGTCTTTGGC USDCR: AGCTCGTGCGTCTCGGTGGTG

The size of PCR amplification product of the wild-type virus was 6286bp, the size of PCR amplification fragment of gI/gE/US9/US2 (i.e.gI/gE/11K/28K) segment-deleted virus was 1960 bp.

PCR assay result confirmed that ORF of gI/gE/US9/US2 (i.e.gI/gE/11K/28K) segment had been completely missing.

EXAMPLE 2 Preparation of PRV HN1201 Strain With Deletion ofgI/gE/11K/28K/TK 2.1 Construction of a Transfer Vector for RecombinantPRV HN1201GFP Virus

According to the sequence of TK gene to be deleted, the homologous armsat its two ends were designed, called TKA and TKB, respectively. TKA andTKB were cloned into pUC19 vector and named pUCTKAB. Then GFP gene wascloned into pUCTKAB, to obtain a transfer vector for recombinant viruswhich was called pUCTKA-GFP-B. The homologous arms in the transfervector are sequences of two sides of TK, therefore the recombinant virusobtained after recombination, was TK gene deleted. FIG. 4 shows thelocation of homologous arms, TKA and TKB in the genome.

2.1.1 Amplification and Cloning of the Homologous Recombinant Arms2.1.1.1 Design of Primers and Preparation of Template

Two pairs of primers were designed for amplifying the homologous arms attwo sides of TK gene according to the gene sequence of HN1201 virus:

The upstream and downstream primers for the homologous arm TKA at theleft side are, respectively:

TKAF: CCGGAATTCGTAGTGCCGGTTGCCCACGTACA(the underline portion refers to the EcoR I cutting site) TKAR:CTAGTCTAGAataacttcgtatagtacacattatacgaagttat CGCTCAGGCTGCCGTTCTGC(the underline portion refers to the Xba Icutting site, lowercase letters refer to the loxp site)

The upstream and downstream primers for the homologous arm TKB at theright side are, respectively:

TKBF: ACATGCATGCataacttcgtataatgtgtactatacgaagttat AACGACGACGGCGTGGGAGG(the underline portion refers to the SphIcutting site, lowercase letters refer to the loxp site) TKBR:CCCAAGCTTAGGGCGACGGCGAAGAAGAGC(the underline portion refers to the Hind III cutting site)

Vero cells were transfected with PRV HN1201, and part of supernatant washarvested when the cytopathic effect of cells reached to 80%, forextracting genomic DNA of virus by using Geneaid Viral Nucleic AcidExtraction kit as the template for amplification of the homologous arms.

2.1.1.2 Amplification and Cloning of the Homologous Arms, TKA and TKB

TKA and TKB were amplified through PCR method by using TAKARA PrimeSTAR,of which the system and condition is as follows:

PRV HN1201 DNA 1 μL PrimeSTAR 0.5 μL 2*primeSTAR GC buffer 25 μL dNTP(25mM) 4 μL Upstream primer 0.5 μL downstream primer 0.5 μL Water Used foradjusting to a final volume of 50 μL

98° C. 2 min 98° C. 10 s 68° C. 1 min } 30 cycles 15 s 68° C. 5 min

TKA and TKB fragments amplified by PCR were separated by electrophoresison agarose gel, and the target fragments were recovered with TIANGEN GelRecovery Kit. TKA fragment and pUC19 vector was digested with both ofEcoR I and XbaI, and the target fragments were recovered, linked by T4DNA ligase, and the product transformed into DH5α. The transformationmix was spread onto plates containing ampicillin, and incubated at 37°C. overnight. A single colony was picked to extract the plasmid and theplasmid was identified after enzyme digestion, and the correct plasmidafter identification was named pUCTKA. pUCTKA and TKB was digested withboth of SalI and HindIII, and the target fragments were recovered,linked by T4 DNA ligase, and the product transformed into DH5α. Thetransformation mix was spread onto plates containing ampicillin, andincubated at 37° C. overnight. A single colony was picked to extract theplasmid and the plasmid was identified by sequencing after enzymedigestion, and the correct plasmid after identification was namedpUCTKAB.

2.1.2 Amplification of Label Gene GFP 2.1.2.1 Removal of MultipleCloning Site (MCS) of GFP Vector pAcGFP-C1

The pAcGFP-C1 plasmid (purchased from Clontech, Catalog No. 632470) wasdigested with Bgl II and Sma I, and the linearized vector was recovered,linked by T4 DNA Ligase after filling-in with DNA Polymerase I Large(Klenow) Fragment, and the product was transformed into the competentcell DH5α to obtain MCS deleted GFP plasmid, named pAcGFPΔMCS.

2.1.2.2 Amplification of GFP Gene

The primers for amplifying GFP were designed according to the sequenceof pAcGFP-C1 vector.

Upstream primer CMVU: ACGCGTCGACTAGTTATTAATAGTAATCAATTACG(the underline portion refers to the SalI cutting site.)Downstream primer SV40R: ACATGCATGCCTAGAATGCAGTGAAAAAAATGC((the underline portion refers to the Sph I cutting site.)

GFP gene was amplified with the template of pAcGFPΔMCS plasmid, of whichthe system and condition is as follows:

pAcGFPΔMCS 1 μL PrimeSTAR 0.5 μL 2*primeSTAR GC buffer 25 μL dNTP(25 mM)4 μL Upstream primer CMVU 0.5 μL Downstream primer SV40R 0.5 μL WaterUsed for adjusting to a final volume of 50 μL

94° C. 2 min 94° C. 30 s 60° C. 30 s 72° C. 2 min 30 cycles {closeoversize brace} 72° C. 5 min

A target band was recovered by electrophoresis on agarose gel forfurther linking.

2.1.3 Linking of GFP Label Gene and pUCTKAB

GFP was digested with both of Sal and Sph I, and the target fragmentswere recovered, linked to pUCTKAB plasmid which had been through thesame double enzyme digestion, and the linked product was transformedinto the competent cell DH5α. The transformation mix was spread ontoplates containing ampicillin, and incubated at 37° C. overnight. Asingle colony was picked to extract the plasmid and the plasmid wasidentified by sequencing after enzyme digestion, and the correct plasmidafter identification was named pUCTKA-GFP-B.

2.2 Acquisition of Recombinant Virus Containing GFP 2.2.1 Acquisition ofRecombinant Virus Through Co-Transfection of Vero Cells With theTransfer Vector and vPRV-gI-gE-11K-28K-DNA

Co-transfection of vero cells was conducted by using lipofectintechnique, wherein 3 μg vPRV-gI-gE-11K-28K− viral genomic DNA and 5 μgthe transfer vector pUCTKA-GFP-B was transfected, in accordance withprocedures of Lipofectamine 2000 Protocol (Invitrogen, Catalog No.11668030). Cells were incubated at 37 ° C. in an incubator containing 5%CO₂. The supernatant of cell culture, i.e. PO recombinant virus, namedrPRV-GFP-gI-gE-11K-28K-TK−, was collected 36-48 h after transfection, oruntil the cytopathic effect was visible and infected cells exhibitedfluorescence.

2.2.2 Plaque Purification of Recombinant VirusrPRV-GFP-gI-gE-11K-28K-TK−

When infected with the obtained PO recombinant virusrPRV-GFP-gI-gE-11K-28K-TK−, vero cells infected were covered with 2%agarose with low melting point. After 48 h when the cytopathic effectbecame apparent and infected cells exhibited obvious fluorescence, aplaque with a green fluorescence was picked and freeze-thawed 3 times in−70 ° C., inoculated at 10-fold serial dilutions into vero cellspreviously laid in six-well plates. Such plaque with a greenfluorescence was continued to be picked for purification. After 11rounds of plaques purification, a purified recombinant virusrPRV-GFP-gI-gE-11K-28K-TK− which was free of PRV-gI-gE-11K-28K-TK− andwith deletion of five genes was obtained.

2.3 Deletion of GFP Label Gene in gI/gE/11K/28K/TK Deleted RecombinantVirus

The pBS185 plasmid expressing Cre enzyme (purchased from addgene, Creenzyme recognizes mutated loxP sites at downstream of TKA and upstreamof TKB, wherein TKA and TKB are homology arms, and deletes sequencebetween two loxp sites) and genomic DNA of recombinant virusrPRV-GFP-gI-gE-11K-28K-TK− was co-transfected into vero cells, with theresults showing relatively obvious cytopathic effect and more singlefluorescence 24 h after transfection. After serial dilution, P0 virusharvested was inoculated for plaque screening; fluorescence-negativeplaque was picked for the next round of purification. After 2 rounds ofscreening and purification, a fluorescence-negative virus was obtained,and named PRV-gI-gE-11K-28K-TK−. PCR identification result afterextraction and purification of viral genomic DNA, showed deletion of TKgene, and also indicated that GFP label gene had been deleted. Theresult showed a successful purification of gI-gE-11K-28K-TK− deletedvirus containing no GFP label gene.

2.4 Confirmation of PRV HN1201 Strain With Deletion of gI/gE/11K/28K/TK

The primers used for identifying deletion of gI/gE/11K/28K were the sameas above.

The viral genome of gI/gE/11K/28K/TK-deleted virus and wild-type virus,was extracted and identified by PCR, with the following primers:

-   TKDCF: cctacggcaccggcaagagca-   TKDCR: cgcccagcgtcacgttgaagac

The size of PCR amplification product of the wild-type virus was 1566bp, the size of PCR amplification fragment of TK deleted virus was 742bp (refer to FIG. 5).

EXAMPLE 3 Preparation of PRV HN1201 Strain With Deletion of gI/gE

PRV HN1201 strain with deletion of gI/gE was prepared by reference tothe method in Example 1 of CN103756977A.

EXAMPLE 4 Pathogenicity Test of Gene-Deleted PRV Strain

25 7-day-old piglets which were negative for pseudorabies antibodies andpseudorabies antigen were randomly divided into 5 groups (A, B, C, D andblank control group), each with 5 piglets. Grouping conditions andchallenge conditions are shown in Table 1.

TABLE 1 Grouping of animals in the pathogenicity test Group Strain usedfor inoculation Dose A PRV HN1201 strain with inoculated with 1 mldeletion of gI/gE/11K/28K (10^(7.0) TCID₅₀/ml)/piglet by prepared inExample 1 intranasal instillation B PRV HN1201 strain with inoculatedwith 1 ml deletion of (10^(7.0) TCID₅₀/ml)/piglet by gI/gE/11K/28K/TKintranasal instillation prepared in Example 2 C PRV HN1201 strain withinoculated with 1 ml deletion of gI/gE prepared (10^(7.0)TCID₅₀/ml)/piglet by in Example 3 intranasal instillation D PRV HN1201strain inoculated with 1 ml (10^(7.0) TCID₅₀/ml)/piglet by intranasalinstillation Blank DMEM medium inoculated with 1 ml/piglet control byintranasal instillation

After inoculation of virus, the temperature of piglets was determineddaily, and clinical signs and death status were observed. The resultsare shown in Table 2.

TABLE 2 Pathogenicity of different genes-deleted PRV HN1201 strains in7-day-old piglets Group Number Clinical signs Death status A A1 Normalbody temperature, no clinical Survived signs A2 Body temperatureincreased for 1 day, Survived no clinical signs A3 Normal bodytemperature, no clinical Survived signs A4 Normal body temperature, noclinical Survived signs A5 Body temperature increased for 1 day,Survived no clinical signs B B1 Normal body temperature, no clinicalSurvived signs B2 Normal body temperature, no clinical Survived signs B3Normal body temperature, no clinical Survived signs B4 Normal bodytemperature, no clinical Survived signs B5 Normal body temperature, noclinical Survived signs C C1 Body temperature increased for 1 day,Survived slightly depression, loss of appetite C2 Body temperatureincreased for 1 day, Survived slightly depression, loss of appetite C3Body temperature increased for 1 day, Survived slightly depression, lossof appetite C4 Body temperature increased for 1 day, Survived slightlydepression, loss of appetite C5 Body temperature increased for 1 day,Survived slightly depression, loss of appetite D D1 Body temperatureincreased for 3 days, Died on day depression, completely loss of 3 afterappetite, neurological signs such as challenge staying lying, dyspnea,trembling, convulsions, turning around, and making strokes with theirarms D2 Body temperature increased for 4 days, Died on day depression,completely loss of 4 after appetite, staying lying, dyspnea, challengetrembling and convulsions. D3 Body temperature increased for 4 days,Died on day depression, completely loss of 4 after appetite,neurological signs such as challenge staying lying, dyspnea, trembling,convulsions, turning around, and making strokes with their arms D4 Bodytemperature increased for 4 days, Died on day depression, completelyloss of 4 after appetite, neurological signs such as challenge stayinglying, dyspnea, trembling, convulsions, turning around, and makingstrokes with their arms D5 Body temperature increased for 4 days, Diedon day depression, completely loss of 4 after appetite, neurologicalsigns such as challenge staying lying, dyspnea, trembling, convulsions,and making strokes with their arms Blank K1 Normal Survived control K2Normal Survived K3 Normal Survived K4 Normal Survived K5 Normal Survived

It showed in the results that inoculation with PRV HN1201 strain in7-day-old piglets could lead to 100% death (5/5) of inoculated piglets,while the virulence of PRV HN1201 strain with deletion of gI/gE/11K/28Kwas significantly decreased, which could only make the temperature of 2piglets increased, without any clinical signs. Inoculation with PRVHN1201 strain with deletion of gI/gE in 7-day-old piglets could stilllead to common clinical signs such as increased body temperature anddepression etc., indicating remaining virulence; while PRV HN1201 strainwith deletion of gI/gE/11K/28K/TK gene had completely lost itsvirulence.

EXAMPLE 5 Preparation of the Live Gene-Deleted PRV Vaccines 5.1Proliferation of Vaccine Virus

The virus seed of PRV HN1201 strain with deletion of gI/gE/11K/28Kprepared in Example 1, PRV HN1201 strain with deletion ofgI/gE/11K/28K/TK prepared in Example 2 and PRV HN1201 strain withdeletion of gI/gE prepared in Example 3 was diluted at 5×10⁴ fold, andthen inoculated into a monolayer of ST cell. After 1 h adhesion, 1000 mlof DMEM medium containing 2% fetal calf serum was added into ST cell,which was then placed at 37° C. in a roller bottle with a rotation speedof 6 rph. The cell medium containing viruses was harvested when thecytopathic effect of cells reached to 80%; the viruses were harvestedafter 2 times of freezing-thawing the medium and the virus titer wasassessed. The virus solution was preserved at low temperature.

5.2 Preparation of a Protective Agent

40 g of sucrose and 8 g of gelatin was added into every 100 ml ofdeionized water, and the solution was autoclaved (under 121° C. for 30min) after fully melted.

5.3 Preparation of Vaccine Virus Suspension

The virus solution prepared and preserved in Example 5.1 was mixed withthe protective agent prepared and preserved in Example 5.2 at a volumeratio of 1:1, distributed into sterilized bottles, each of whichcontaining 2.6 ml and the mixed virus solution was freeze-dried. Thevaccine was tested and determined to be free of contamination ofbacterium and exogenous viruses and the content of virus was consistentwith that before freeze-drying. The batch number of PRV HN1201 strainwith deletion of gI/gE/11K/28K prepared in Example 1, PRV HN1201 strainwith deletion of gI/gE/11K/28K/TK prepared in Example 2 and PRV HN1201strain with deletion of gI/gE prepared in Example 3 were 20140501,20140502 and 20140503, respectively.

EXAMPLE 6 Immunogenicity Assay of the Live Gene-Deleted PRV Vaccines

12 9-day-old piglets which were negative for PRV antibodies and PRVantigens were randomly divided into 5 groups, each with 5 piglets, andthe piglets were injected with the vaccines prepared in Example 5according to Table 3. The vaccine control group was inoculated with thelive PRV vaccine, Bartha K-61strain purchased from HIPRA, Spain, BatchNo. 42RH, at the dosage from the protocol. The blank control group wasinoculated with 1 mL/piglet of DMEM medium. The piglets were challengedwith 1□10^(7.0)TCID₅₀/piglet of PRV HN1201 strain on day 28 afterimmunization. After challenge, the body temperature of piglets wasdetermined daily, and in the meanwhile clinical signs and death statuswere observed (The results are shown in Table 3), the blood of pigletsin all the experimental groups and control groups was collectedrespectively before challenge.

TABLE 3 Grouping of animals in the pathogenicity test Group Vaccinesinjected Dose Group I Batch No. inoculated with 1 ml 20140501 10^(6.0)TCID₅₀/piglet by intramuscular injection Group II Batch No. inoculatedwith 1 ml 20140502 10^(6.0) TCID₅₀/piglet by intramuscular injectionGroup III Batch No. inoculated with 1 ml 20140503 10^(6.0) TCID₅₀/pigletby intramuscular injection Vaccine control Live PRV inoculated with 2 mlgroup 2 vaccine 10^(6.0) TCID₅₀/piglet by intramuscular injection Blankcontrol DMEM inoculated with 1 mL/piglet group medium by intramuscularinjection

The piglets were challenged with 1□10^(7.0)TCID₅₀/piglet (1 ml/piglet)of PRV HN1201 strain on day 28 after immunization. After challenge, thebody temperature of piglets was determined daily, and in the meanwhileclinical signs and death status were observed (The results are shown inTable 5).

TABLE 5 clinical status and challenge status for piglets challengedafter immunization with live PRV vaccines clinical signs and death Groupstatus Rate of protection Group I Normal body temperature, 100% (5/5)normal appetite, no abnormal clinical signs, survived Group II Normalbody temperature, 100% (5/5) normal appetite, no abnormal clinicalsigns, survived Group III After immunization, body 100% (5/5)temperature increased, slightly depression and loss of appetite. Afterchallenge, normal body temperature, normal appetite, no abnormalclinical signs, survived Vaccine Body temperature of three  80% (4/5)control piglets increased for 7-10 group days, depression, loss ofappetite, one died. Blank Body temperature of three  0% (0/5) controlpiglets increased for 7-10 group days, depression in all piglets,partially or completely loss of appetite, significant clinical signs,two piglets died on day 4 after challenge, and all died within 5 daysafter challenge.

The result from Table 5 indicated that immunizing piglets with thegene-deleted PRV vaccines prepared in example 5 can blocked virusinfection (i.e. displaying clinical signs), and provide 100% (5/5)protection rate for piglets, while all the piglets in the blank controlgroup died by day 5 after challenge, therefore the PRV vaccines in threeexperimental groups can provide excellent protection, showing excellentimmune protection and safety; meanwhile it indicated that eitherdeletion of gI/gE/11K/28K or deletion of gI/gE/11K/28K/TK for PRV strainwould not affect the immunogenicity. For the vaccine group with onlydeletion of gI/gE, the clinical signs such as increased body temperaturecould not be avoided, while the vaccine still possessed goodimmunogenicity. Whereas the commercial vaccines in the prior art cannotprovide a full protection to pigs.

EXAMPLE 7 Construction of Gene-Deleted Variant Strains of NVDC-PRV-BJStrain, NVDCPRV-HEB Strain and NVDC-PRV-SD Strain, HN1202 PRV VariantStrain

gI/gE/11K/28K genes and gI/gE/11K/28K/TK genes were deleted from theparent strains, NVDC-PRV-BJ strain, NVDC-PRV-HEB strain and NVDC-PRV-SDstrain (Xiuling Yu, Zhi Zhou, Dongmei Hu,et al. Pathogenic PseudorabiesVirus, China, 2012 Emerging Infectious Diseases, www.cdc.gov/eid ol. 20,No. 1, January 2014) (the applicant promises to open it to public for 20year from the patent application date according to provisions ofGuidelines for Patent Examination), HN1202 strain (deposited in theChina Center for Type Culture Collection on Aug. 26, 2013, of which theaccession number is CCTCC NO. V 201335 and the address is WuhanUniversity, Wuhan, China), according to methods in Example 1 and 2. Thenames of the attenuated strains obtained were NVDC-PRV-BJ with deletionof gI/gE/11K/28K/TK, NVDCPRV-HEB with deletion of gI/gE/11K/28K/TK,NVDC-PRV-SD with deletion of gI/gE/11K/28K/TK, and PRVHN1202 withdeletion of gI/gE/11K/28K/TK. The deletion of genes was verified throughcomparison of PCR results with that of parent strains respectively.

EXAMPLE 8 Preparation of Vaccine Compositions of the Attenuated VariantStrains of NVDC-PRV-BJ Strain, NVDC-PRV-HEB Strain and NVDC-PRV-SDStrain, HN1202 PRV Strain

Each attenuated vaccine strains prepared in Example 7 was proliferatedaccording to the method from Example 5.1, mixed with the protectiveagent (prepared by adding 40 g of sucrose and 8 g of gelatin into every100 ml of deionized water, and autoclaved (under 121° C. for 30 min)after fully melted) at a volume ratio of 1:1 and the mixed vaccinecompositions were freeze-dried. The batch numbers of NVDC-PRV-BJ strainwith deletion of gI/gE/11K/28K/TK, NVDCPRV-HEB strain with deletion ofgI/gE/11K/28K/TK, NVDC-PRV-SD strain with deletion of gI/gE/11K/28K/TKand PRV HN1201 strain with deletion of gI/gE/11K/28K/TK were Q01, Q02,Q03 and Q04, respectively.

EXAMPLE 9 Pathogenicity Test of the Virus Strains Prepared in Example 7

Pathogenicity test was conducted according to the method in Example 4,in which the piglets were randomly divided into 5 groups, each with 5piglets, inoculated with 1 ml (10^(7.0)TCID₅₀/ml) of NVDC-PRV-BJ strainwith deletion of gI/gE/11K/28K/TK, NVDC-PRV-HEB strain with deletion ofgI/gE/11K/28K/TK, NVDC-PRV-SD strain with deletion of gI/gE/11K/28K/TK,and PRV HN1202 strain with deletion of gI/gE/11K/28K/TK by intranasalinstillation, respectively. The results showed that all the piglets werealive in each group, with normal body temperature and no clinical signs.It proved that the virulence of mutated PRV strain was reduced throughdeletion of gI/gE/11K/28K/TK genes.

EXAMPLE 10 Immunogenicity Assay of the Vaccines Prepared in Example 8

Immunogenicity assay of the vaccines prepared in Example 8 was conductedaccording to the method and dose in Example 6, in the meanwhile thepiglets in the vaccine control group were inoculated with the live PRVvaccine, HB-98 strain Batch No. 1308011-1 (purchased from China AnimalHusbandry Industry Co., Ltd. Chengdu Medical Equipments Factory). Thepiglets were challenged with 1□10^(7.0)TCID₅₀/piglet of PRV HN1201strain on day 28 after immunization. After challenge, the bodytemperature of piglets was determined daily, and in the meanwhileclinical signs and death status were observed (the results are shown inTable 6).

TABLE 6 clinical status and challenge status for piglets challengedafter immunization with live PRV vaccines clinical signs and death Rateof Group Vaccines status protection Group IV Q01 Normal body 100% (5/5)temperature, normal appetite, no abnormal clinical signs, survived GroupV Q02 Normal body 100% (5/5) temperature, normal appetite, no abnormalclinical signs, survived Group VI Q03 Normal body 100% (5/5)temperature, normal appetite, no abnormal clinical signs, survived GroupVII Q04 Normal body 100% (5/5) temperature, normal appetite, no abnormalclinical signs, survived Vaccine the live PRV Body temperature of five 80% (4/5) control vaccine, HB- piglets increased for 7-10 group 98strain days, loss of appetite, one Batch No. piglet died and four1308011-1 survived. Blank DMEM Body temperature of all  0% (0/5) controlmedium piglets increased, group depression in all piglets, partially orcompletely loss of appetite, significant clinical signs, two pigletsdied on day 4 after challenge, and all died within 5 days afterchallenge.

The result from Table 6 indicated that immunizing piglets with the PRVvaccines prepared in Example 8 can block virus infection (i.e.displaying clinical signs), and provide 100% (5/5) protection rate forpiglets, while the vaccine control group can only provide 80% (4/5)protection rate for piglets, and all the piglets in the blank controlgroup died by day 5 after challenge, therefore the PRV vaccines of thepresent invention can provide excellent protection. In addition, thepiglets exhibited substantially no clinical signs, indicating excellentimmune protection of the PRV vaccines relative to live vaccines in theprior art.

EXAMPLE 11 Monitoring of gB Antibodies After Immunization With DifferentStrain Vaccines

15 piglets at the age of around 13 days which were negative for PRVantigens and PRV antibodies were randomly divided into 5 groups, eachwith 5 piglets. Groups 1-3 were injected with the vaccine prepared inExample 5,which is PRV HN1201 strain with deletion of gI/gE/11K/28K/TK,with Batch No. 20140502, the live PRV vaccine Bartha K-61 strain, withBatch No. 66KR, purchased from HIPRA, Spain, and the live PRV vaccine,K-61, with Batch No. 195-B59B purchased from Boehringer Ingelheim (US)respectively. All the dose for immunization is 1 ml/piglet (forcommercial vaccine, 1 piglet dosage/piglet, according to protocols; thePRV HN1201 with deletion of gI/gE/11K/28K/TK vaccine,10^(6.0)TCID₅₀/piglet). The blank control group was inoculated with 1mL/piglet of DMEM medium. The blood of piglets was collected on day 8,10, 12, 14 and 21 after immunization, and gB antibody was determinedaccording to the protocol of gB ELISA antibody detection kit (purchasedfrom Biochek, Batch No. F S5763, Expiry Date: Jan. 7, 2015) after theserum was separated. The detailed results of detection are shown inTable 7 below.

TABLE 7 Results of detection of gB antibodies of piglets afterimmunization. Before Day 8 after Day 10 after immunization immunizationimmunization Group No. of piglet OD405nm S/P OD405nm S/P OD405nm S/P PRVHN1201 1# 0.184 0.025 0.398 0.469 0.439 0.555 strain with 2# 0.170−0.004 0.369 0.409 0.453 0.584 deletion of 3# 0.172 0.000 0.263 0.1890.360 0.390 gI/gE/11K/28K/ 4# 0.181 0.019 0.320 0.307 0.494 0.669 TKvaccine 5# 0.182 0.021 0.339 0.347 0.400 0.474 with Batch No. 20140502Bartha K-61 6# 0.177 0.010 0.223 0.106 0.243 0.147 7# 0.176 0.008 0.2560.174 0.286 0.237 8# 0.167 −0.010 0.224 0.108 0.246 0.154 9# 0.186 0.0290.221 0.102 0.219 0.098 10# 0.175 0.006 0.242 0.145 0.277 0.218 K-61 11#0.162 −0.019 0.195 0.059 0.185 0.035 12# 0.16 −0.023 0.174 0.009 0.1920.052 13# 0.167 −0.007 0.182 0.028 0.218 0.113 14# 0.16 −0.023 0.1990.068 0.201 0.073 15# 0.17 0.000 0.219 0.115 0.225 0.129 Day 12 afterDay 14 after Day 21 after immunization immunization immunization GroupNo. of piglet OD405nm S/P OD405nm S/P OD405nm S/P PRV HN1201 1# 0.4710.621 0.678 1.051 1.069 1.863 strain with 2# 0.510 0.702 0.631 0.9530.984 1.686 deletion of 3# 0.453 0.584 0.496 0.673 0.619 0.928gI/gE/11K/28K/ 4# 0.596 0.881 0.687 1.070 0.844 1.396 TK vaccine, 5#0.602 0.893 0.547 0.779 0.690 1.076 Batch No. 20140502 Bartha K-61 6#0.275 0.214 0.290 0.245 0.570 0.827 7# 0.302 0.270 0.317 0.301 0.4180.511 8# 0.283 0.231 0.309 0.285 0.315 0.297 9# 0.211 0.081 0.223 0.1060.316 0.299 10# 0.272 0.208 0.299 0.264 0.486 0.652 K-61 11# 0.239 0.1620.274 0.244 0.314 0.338 12# 0.205 0.082 0.211 0.096 0.277 0.251 13#0.248 0.183 0.25 0.188 0.449 0.655 14# 0.256 0.202 0.285 0.27 0.3210.354 15# 0.28 0.258 0.3 0.305 0.385 0.505 Note: evaluation criteria:negative, S/P value ≦0.499; positive, S/P value ≧0.500.

In conclusion, the antibody test results showed that, all gB antibodiesturned positive on day 12 after immunization with PRV HN1201 strain withdeletion of gI/gE/11K/28K/TK, while not all the gB antibodies had turnedpositive on day 21 after immunization with the two control vaccine. Itshowed that PRV HN1201 strain with deletion of gI/gE/11K/28K/TK couldprovide earlier immune protection.

EXAMPLE 12 Monitoring of gE Antibodies After Immunization With FourGenes Deleted Strain Vaccine and Challenge

15 piglets at the age of around 13 days which were negative for PRVantigens and PRV antibodies were randomly divided into 3 groups, eachwith 5 piglets. Groups 1-3 were injected with the vaccine prepared inExample 5,which is PRV HN1201 strain with deletion of gI/gE/11K/28K/TK,with Batch No. 20140502, the live PRV vaccine, Bartha K-61 strain, withBatch No. 66KR, purchased from HIPRA, Spain, and the live PRV vaccine,K-61, with Batch No. 195-B59B purchased from Boehringer Ingelheim (US).All the dose for immunization is 1 ml/piglet (for commercial vaccine, 1piglet dosage/piglet, according to protocols; the PRV HN1201 withdeletion of gI/gE/11K/28K/TK vaccine, 10^(6.0)TCID₅₀/piglet). Thepiglets were challenged with 10^(7.0)TCID₅₀/piglet, 1 ml/piglet of PRVHN1201 strain on day 21 after immunization. The blood of piglets wascollected daily continuously from day 7 to day 14 after challenge, andgE antibody was determined according to the protocol of gE ELISAantibody detection kit (purchased from IDEXX Co., Batch No. AK650,Expiry Date: Jun. 13, 2015) after the serum was separated. The resultsshowed that gE antibody was still negative (If the value of S/N is lessor equal to 0.60, the sample should be determined as PRV gE antibodypositive) on Day 14 after challenge when the piglets were immunized withthe vaccine prepared in Example 5, PRV HN1201 with deletion ofgI/gE/11K/28K/TK with Batch No. 20140502, while gE antibody becamepositive at different level when the piglets were immunized with the twocommercial vaccines. The detailed results of deletion are shown in Table8 below.

TABLE 8 Results of detection of gE antibody of piglets afterimmunization. No Before Day 7 after Day 8 after Day 9 after Dayb10 afterof challenge challenge challenge challenge challenge Group pigletOD650nm S/N OD650nm S/N OD650nm S/N OD650nm S/N OD650nm S/N PRV 1# 1.0241.041 0.917 0.932 0.956 0.972 0.860 0.874 0.863 0.877 HN1201 2# 1.0061.008 0.979 0.980 0.931 0.932 0.889 0.890 0.780 0.781 with 3# 1.0701.072 0.990 0.991 1.007 1.009 0.970 0.971 0.929 0.930 deletion 4# 1.0521.054 0.795 0.796 0.899 0.872 0.972 0.943 1.000 0.970 of 5# 0.969 0.9700.912 0.913 0.915 0.916 0.922 0.923 0.683 0.684 gI/gE/11K/ 28K/TK,vaccine with Batch No. 20140502 Bartha 6# 1.045 1.078 0.634 0.654 0.6840.706 0.587 0.606 0.518 0.535 K-61 7# 1.063 1.097 0.788 0.813 0.7580.782 0.664 0.685 0.612 0.632 8# 1.008 1.040 0.897 0.926 0.857 0.8840.784 0.809 0.783 0.808 9# 1.017 1.050 0.720 0.743 0.637 0.657 0.5990.618 0.467 0.482 10# 0.987 1.019 0.871 0.899 0.701 0.723 0.656 0.6770.655 0.676 K-61 11# 0.905 0.934 0.946 0.976 0.698 0.720 0.643 0.6640.618 0.638 12# 1.024 1.057 0.898 0.927 0.773 0.798 0.688 0.710 0.7600.784 13# 1.030 1.063 0.957 0.928 0.965 0.936 0.913 0.886 0.732 0.71014# 0.963 0.934 0.757 0.734 0.899 0.872 0.972 0.943 1.000 0.970 15#0.944 0.916 0.747 0.725 0.591 0.573 0.543 0.527 0.531 0.515 Day 11 afterDay 12 after Day 13 after Day 14 after No of challenge challengechallenge challenge Group piglet OD650nm S/N OD650nm S/N OD650nm S/NOD650nm S/N PRV 1# 0.884 0.898 0.877 0.891 0.871 0.885 0.880 0.894HN1201 2# 0.854 0.855 0.780 0.781 0.793 0.794 0.732 0.733 with 3# 0.9070.908 0.905 0.906 0.904 0.905 1.067 1.069 deletion 4# 0.965 0.936 0.8640.838 0.997 0.967 0.929 0.901 of 5# 0.623 0.624 0.718 0.719 0.784 0.7850.718 0.719 gI/gE/11K/ 28K/ TK vaccine, with Batch No. 20140502 Bartha6# 0.552 0.570 0.482 0.497 0.463 0.478 0.456 0.471 K-61 7# 0.664 0.6850.533 0.550 0.499 0.515 0.478 0.493 8# 0.749 0.773 0.647 0.668 0.7000.722 0.753 0.777 9# 0.450 0.464 0.410 0.423 0.432 0.446 0.433 0.447 10#0.633 0.653 0.699 0.721 0.684 0.706 0.676 0.698 K-61 11# 0.568 0.5860.472 0.487 0.472 0.487 0.449 0.463 12# 0.745 0.769 0.659 0.680 0.6590.680 0.714 0.737 13# 0.785 0.761 0.678 0.658 0.505 0.490 0.425 0.41214# 0.965 0.936 0.864 0.838 0.997 0.967 0.929 0.901 15# 0.578 0.5610.528 0.512 0.457 0.443 0.398 0.386

The above results indicated that the vaccine strains in the presentinvention has a better immunogenicity than commercial vaccine in theprior art, and after immunization therewith a faster generation of theantibody can be achieved, and the effective amplification of virus inthe body of pigs can be blocked, and gE antibody is negative.

Those are only preferred embodiments of the present invention asdescribed above, which cannot be used to limit the present invention.Any change, substitution or modification etc., which are within thespirit and principle of the invention, should be included within thescope of protection of the present invention.

What is claimed is:
 1. An attenuated strain of porcine pseudorabiesvirus comprising a first attenuated porcine pseudorabies virus strain,wherein the first attenuated strain of porcine pseudorabies comprises aporcine pseudorabies virus strain with inactivation of gI/gE/11K/28Kprotein.
 2. The attenuated strain of porcine pseudorabies virus of claim1, wherein the first attenuated porcine pseudorabies virus strain is avariant strain of pseudorabies virus.
 3. The attenuated strain ofporcine pseudorabies virus of claim 1, wherein a whole ORF is deletedfrom a gI/gE/11K/28K gene of the first attenuated porcine pseudorabiesvirus strain.
 4. The attenuated strain of porcine pseudorabies virus ofclaim 2, wherein the variant strain of pseudorabies virus comprises avirus strain in which gE protein has 95% or greater homology with SEQ IDNO.
 05. 5. The attenuated strain of porcine pseudorabies virus of claim4, wherein said variant strain of pseudorabies virus is obtained throughisolation, and when infection with said variant strain occurs in pigspreviously immunized with a second attenuated gene deleted porcinepseudorabies virus strain, wherein the second attenuated gene deletedporcine pseudorabies virus strain is prepared according to the priorart, the pigs display clinical signs of infection selected from thegroup consisting of high fever, depression, and partial or complete lossof appetite.
 6. The attenuated strain of porcine pseudorabies virus ofclaim 5, wherein when exposed to said variant strain of pseudorabiesvirus, pigs previously immunized with the second attenuated porcinepseudorabies virus strain develop a pseudorabies infection, and whereinthe second attenuated porcine pseudorabies vaccine is a prior art strainwith deletion of one or more of the group consisting of gE, TK and gIgenes.
 7. The attenuated strain of claim 6, wherein the pseudorabiesinfection comprises clinical signs of infection selected from the groupconsisting of depression and loss of appetite among piglets at the ageof 9-10 days.
 8. The attenuated strain of porcine pseudorabies virus ofclaim 1, wherein the first porcine pseudorabies virus strain is selectedfrom the group consisting of an HN1201 strain, HN1202 strain, JS-2012strain, PRV HeN1 strain, NVDC-PRV-BJ strain, NVDCPRV-HEB strain orNVDC-PRV-SD strain, PRV TJ strain, and PRV-ZJ01 strain.
 9. Theattenuated strain of porcine pseudorabies virus of claim 1, wherein thefirst attenuated strain of porcine pseudorabies virus strain comprises agene variation resulting in inactivation of TK protein.
 10. Theattenuated strain of porcine pseudorabies virus of claim 9, wherein thegene variation comprises a nucleotide sequence located at a sitecorresponding to a TK gene of the first attenuated porcine pseudorabiesvirus strain, and wherein the nucleotide sequence encodes for SEQ ID NO.4.
 11. A vaccine composition, comprising an immunizing amount of anantigen of an attenuated strain of porcine pseudorabies and a carrier,wherein the attenuated strain of porcine pseudorabies comprises agenetic variation resulting in inactivation of gI/gE/11K/28K protein.12. The vaccine composition of claim 11, wherein the immunizing amountcomprises at least 10^(6.0)TCID₅₀/ml of the attenuated strain of porcinepseudorabies virus.
 13. The vaccine composition of claim 11, wherein theantigen comprises a live attenuated strain of porcine pseudorabiesvirus, and wherein the vaccine composition further comprises acryoprotectant.
 14. The vaccine composition of claim 11, furthercomprising an inactivated pathogen or antigen.
 15. The vaccinecomposition of claim 14, wherein the inactivated pathogen or antigen isselected from the group consisting classical swine fever virus, antigenof porcine reproductive and respiratory syndrome virus, antigen ofporcine circovirus, antigen of haemophilus parasuis, and antigen ofmycoplasma.
 16. A method of treating and preventing pseudorabiesinfection, comprising immunizing a pig with an antigen comprising aporcine pseudorabies virus strain with inactivation of gI/gE/11K/28Kprotein.
 17. The method of claim 16, wherein the porcine pseudorabiesvirus strain comprises a virus strain in which gE protein has 95% orgreater homology with SEQ ID NO. 05.